The assembly of influenza A virus involves the specific interaction of several viral components at a predetermined location within the virus-infected cell. The viral proteins involved in this process are key components in the generation of vaccines as well as in the adaption of virus to new hosts but precise roles in the assembly process have yet to be ascribed or identified. The application of reverse genetics techniques and new technologies such as RNA interference allows for the precise dissection of the assembly process and the determination of precise functional domains important for virus assembly to take place. To fully understand the viral factors involved in influenza A virus assembly, a detailed molecular investigation of the domains of the M1 (matrix) and M2 proteins involved in virus assembly will be undertaken. The amino acid sequences in M1 and M2 that are important in targeting the proteins to sites of virus budding will be identified. The mechanism behind the anti-M2 antibody mediated inhibition of influenza A virus budding will be elucidated and specific interactions between the M1 protein and the M2 cytoplasmic tail will be characterized. Finally, the intracellular localization of M1 will be analyzed in cells infected with recombinant viruses bearing mutations in the HA and NA proteins that prevent their association with glycolipid rafts or the apical membrane. Recombinant viruses bearing specific mutations in one or several viral proteins will be generated and characterized for their sensitivity to anti-M2 antibodies, antiviral drug sensitivity and virulence. These studies should shed additional light on the mechanisms responsible for the generation of new, highly virulent influenza A viruses as well as further define the interactions that govern antibody and drug sensitivity.